ThermoGen proposes to develop a regulated expression system for the extreme thermophile Thermus flavus. This system will allow production of commercially important thermostable enzymes. In the Phase I feasibility study we will select promoter regions from Thermus, perform minimal structural analyses, and test their regulation using reporter genes in T. flavus. To fulfill this purpose, we will develop an integrative promoter-probe shuttle vector for Thermus based on a promoterless Thermo-beta-galactosidase and kanamycin phosphotransferase and a chromosomal integration system. Since commercially important proteins are often secretable, a search for signals suitable for regulated expression-secretion system will also be done. Phase II will focus on the development of the expression system into a functional tool for expressing both endogenous enzymes in Thermus, and for model experiments in protein thermostabilization. Further study on expression of hyperthermophilic proteins is also planed. In Phase III the expression system will be applied to the development of commercially important enzymes. PROPOSED COMMERCIAL APPLICATION: ThermoGen will develop an expression system for the extreme thermophile Thermus flavus. This expression system will allow the selective thermostabilization of mesophilic proteins with the emphasis of getting new products with therapeutic and industrial application.